Affinity of the monoclonal antibody M1 directed against the FLAG peptide

The FLAG (Sigma, St. Louis, MO, USA) peptide is a frequently used hydrophilic and immunogenic fusion tag which was specifically designed to facilitate rapid purification by immunoaffinity chromatography. The monoclonal antibody M1 recognizes the free N-terminus of the peptide tag in a calcium dependent manner. Dissociation of the complex can be performed by the addition of chelating agents such as EDTA. This effect can be exploited for immunoaffinity purification of FLAG-tagged fusion proteins. Kinetic information obtained from monitoring interactions in real-time measurement (Biacore 2000) using surface plasmon resonance as detection principle did not show any difference for association and dissociation rate constants in the presence (ka=3.03·103 M−1 s−1, kd=1.25·10−3 s−1) and in the absence of Ca2+ (ka=3.59·103 M−1 s−1, kd=1.16·10−3 s−1). These findings corroborate the reports from Mol. Immunol. 33 (1996) 601–608 describing similar binding analyzed by enzyme-linked immunosorbent assay experiments. These investigations are in contrast to the observations in immunoaffinity chromatography with immobilized anti-FLAG antibody M1.